Microscopy and, in particular, fluorescence microscopy plays an ever increasing role as a tool to improve the understanding of cellular processes such as receptor clustering and trafficking. The present application originated from a number of immunological studies which are being carried out by the co-PI. These studies relate to questions of recognition by the T cell receptor in autoimmune disease models (experimental autoimmune encephalomyelitis (EAE); collagen induced arthritis (CIA) and trafficking of the MHC class I-related receptor, FcRn. The overall objective of this multidisciplinary bioengineering project is to provide important additional tools for the analysis of experimental results obtained using fluorescence microscopy, with the emphasis on tools for three dimensional image sets. The premise of the application is that in a number of respects the analysis capabilities lag behind the recent developments in hardware and sample preparation methodology. The proposed study will investigate new methods and approaches to various problems in immunology and cellular biology. All proposed approaches will be rigorously analyzed with simulated and experimental data. Our specific aims are: first, to investigate methods for quantitative analysis of clustering and co-localization. Second, deconvolution algorithms and several modifications will be analyzed. Third, a software suite will be written for acquisition and analysis of fluorescence microscopy images. The capabilities of this package will not only include the necessary functionality to carry out the earlier specific aims, but will provide a powerful development environment for advanced image analysis for microscopy.